The Melan-A/MART-1 gene product is frequently recognized by tumor-specific HLA-A2-restricted CTL. An immunodominant nonapeptide has been localized to the region spanning residues 27-35. However, the decapeptide including residues 26-35 (the nonapeptide extended NH2 terminally by one residue) appeared to be recognized as efficiently as the nonapeptide. In this study, we show that the optimal length immunodominant peptide appears to correspond to the decapeptide 26-35, as assessed by quantitative analyses of both 4 polyclonal and 13 monoclonal populations of specific CTL. Functional assays of peptide binding to HLA-A2 indicate that the decapeptide is significantly a more efficient binder than the nonapeptide. Moreover, analogues of the decapeptide including substitutions at a secondary HLA-A2 peptide anchor further improve decapeptide binding. Finally, we show that the functional (9 CTL clones analyzed) and structural TCR repertoire (7 CTL clones) of a group of specific CTL clones is rather diverse. The findings reported here may have important implications for future peptide-based melanoma vaccination trials as well as for the monitoring of specific CTL responses in vivo.

Conference Article| May 01 1997 Antigens recognized by T-lymphocytes on human tumours P. G. Coulie; P. G. Coulie * *To whom correspondence should be addressed. Search for other works by this author on: This Site PubMed Google Scholar B. J. Van den Eynde; B. J. Van den Eynde 1Cellular Genetics Unit, Université Catholique de Louvain, Brussels, Belgium and Ludwig Institute for Cancer Research, B1200 Brussels, Belgium Search for other works by this author on: This Site PubMed Google Scholar P. van der Bruggen; P. van der Bruggen 1Cellular Genetics Unit, Université Catholique de Louvain, Brussels, Belgium and Ludwig Institute for Cancer Research, B1200 Brussels, Belgium Search for other works by this author on: This Site PubMed Google Scholar A. Van Pel; A. Van Pel 1Cellular Genetics Unit, Université Catholique de Louvain, Brussels, Belgium and Ludwig Institute for Cancer Research, B1200 Brussels, Belgium Search for other works by this author on: This Site PubMed Google Scholar T. Boon T. Boon 1Cellular Genetics Unit, Université Catholique de Louvain, Brussels, Belgium and Ludwig Institute for Cancer Research, B1200 Brussels, Belgium Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1997) 25 (2): 544–548. https://doi.org/10.1042/bst0250544 Article history Received: October 29 1996 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation P. G. Coulie, B. J. Van den Eynde, P. van der Bruggen, A. Van Pel, T. Boon; Antigens recognized by T-lymphocytes on human tumours. Biochem Soc Trans 1 May 1997; 25 (2): 544–548. doi: https://doi.org/10.1042/bst0250544 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: CTL, cytolytic T-lymphocyte © 1997 Biochemical Society1997 Article PDF first page preview Close Modal You do not currently have access to this content.

A cytolytic T lymphocyte (CTL) clone that lyses many HLA-A2 melanomas was derived from a population of tumor-infiltrating lymphocytes of an HLA-A2 melanoma patient. The gene coding for the antigen recognized by this CTL was identified by transfection of a cDNA library. It is the gene which has been reported to code for N-acetylglucosaminyltransferase V (GnT-V). Remarkably, the antigenic peptide recognized by the CTL is encoded by a sequence located in an intron. In contrast to the fully spliced GnT-V mRNA, which was found in a wide range of normal and tumoral tissues, the mRNA containing the intron region coding for the antigen was not found at a significant level in normal tissues. This mRNA was observed to be present in about 50% of melanomas. Our results suggest that a promoter located near the end of the relevant intron is activated in melanoma cells, resulting in the production of an mRNA coding for the antigen.

T lymphocytes recognize antigens consisting of peptides presented by class I and II major histocompatibility complex (MHC) molecules. The peptides identified so far have been predictable from the amino acid sequences of proteins. We have identified the natural peptide target of a CTL clone that recognizes the tyrosinase gene product on melanoma cells. The peptide results from posttranslational conversion of asparagine to aspartic acid. This change is of central importance for peptide recognition by melanoma-specific T cells, but has no impact on peptide binding to the MHC molecule. This posttranslational modification has not been previously described for any MHC-associated peptide and represents the first demonstration of posttranslational modification of a naturally processed class I-associated peptide. This observation is relevant to the identification and prediction of potential peptide antigens. The most likely mechanism for production of this peptide leads to the suggestion that antigenic peptides can be derived from proteins that are translated into the endoplasmic reticulum.

The human tyrosinase gene has been reported previously to code for two distinct antigens recognized on HLA‐A2 melanoma cells by autologous cytolytic T lymphocytes (CTL). By stimulating lymphocytes of melanoma patient MZ2 with a subclone of the tumor cell line of this patient, we obtained a CTL clone that lysed this subclone but did not lyse other subclones of the same melanoma cell line. The sensitive melanoma subclone was found to express a much higher level of tyrosinase than the others, suggesting that the antigen recognized by the CTL might be encoded by tyrosinase. Transfection of a tyrosinase cDNA demonstrated that the CTL clone indeed recognized a tyrosinase product presented by HLA‐B*4403. The relevant antigenic peptide corresponds to residues 192–200 of the tyrosinase protein. Lymphoblastoid cells of the B*4402 subtype were not recognized by the CTL following incubation with the peptide. Nevertheless, by stimulating in vitro lymphocytes of a healthy HLA‐B*4402 donor with autologous adherent cells pulsed with the same peptide, we obtained a CTL clone which recognized tumor cells expressing tyrosinase and HLA‐B*4402. As HLA‐B44 is expressed in 24% of Caucasians, the tyrosinase‐B44 antigen may constitute a useful target for specific immunotherapy of melanoma.

The requirements for generating CD8+ CTLs against the mastocytoma P815 were first defined by using an allogeneic mixed lymphocyte tumor culture (MLTC). Both the expansion of effector lymphocytes and the acquisition of lytic activity were dependent on the presence of accessory cells, but not CD4+ lymphocytes. Several factors were examined for their ability to replace accessory cell function. Expression of B7-1 by P815 was sufficient to induce IL-2 production by CD8+ cells, but substantial proliferation was achieved only if IL-6 was provided as well. Although IL-12 had little effect on the net proliferation of developing effector cells, it increased specific lytic activity 10-fold, acted synergistically with B7-1 in induction of IFN-gamma production during primary stimulation, and resulted in a shift toward a Th1 cytokine profile following secondary stimulation. These costimulatory factors were then studied in a primary syngeneic MLTC by using splenocytes from nonimmunized DBA/2 mice, an approach that had never before succeeded in generating specific CTLs. The combination of B7-1, IL-6, and IL-12 was sufficient to induce P815-specific CTL activity after a 5-day MLTC, which was expanded optimally following secondary stimulation in the presence of B7-1, IL-2, and IL-7. The irrelevant syngeneic tumor L1210, constructed to express B7-1 and the P815-derived tumor Ag gene P1A, also generated CTLs that lysed the parental P815. The combination of B7-1, IL-6, and IL-12 should be useful in the derivation of other tumor-specific CTLs in vitro, and may constitute the optimal stimuli for rapid proliferation and differentiation of helper-independent, tumor-specific CTLs in vivo.

Delayed-type hypersensitivity (DTH) responses, mediated by CD8(+) cells and detected by skin test assay, occur in sensitized mice in response to challenge with class I-restricted antigenic peptides of mutagenized (tum(-)) P815 mastocytoma cells. In contrast, a nonapeptide related to a tumor rejection antigen, P815AB, failed in this study to elicit DTH after sensitization of mice with irradiated tumor cells or adoptive transfer of P815AB-pulsed dendritic cells. Unresponsiveness, however, could be overcome by immunization with tumor cells co-expressing P815AB and tum(-) antigens. When used for cell pulsing in vitro, a mixture of P815AB and tum(-) peptides was also highly effective in inducing anti-P815AB reactivity, as was the combined use of P815AB and class II-restricted peptides of tetanus toxin or Plasmodium berghei circumsporozoite protein. While the effector phase of the CD8(+) cell-mediated DTH to P815AB was unaffected by the ablation of CD4(+) cells, the same treatment, or neutralization of IFN-gamma, negated the induction of reactivity if it occurred at the time of sensitization. Thus, defective activation of CD4(+) cells may contribute to the poor immunogenicity of P815AB. Besides providing an insight into the mechanisms of anti-tumor protection induced by tum(-) cells, these data offer useful information for the design of vaccination strategies against identified tumor antigens.

We reported previously that the mouse tumor P815 expresses four distinct antigens (A, B, C, D) recognized by syngeneic cytolytic T lymphocytes (CTL). A fifth P815 antigen (E) was identified by means of a CTL clone derived from tumor‐infiltrating lymphocytes. We compared a number of mice for the orientation of their CTL response with respect to the various P815 antigens. Lymphocytes from mice inoculated subcutaneously with living P815 cells were stimulated in vitro with tumor cells and the resulting CTL were tested against targets expressing either antigens A and B or antigens C, D and E. Many mice had an asymmetrical response, some producing CTL directed almost exclusively against antigens A, B and others producing CTL directed almost exclusively against C, D. E. When mice were inoculated into two separate sites, different orientations in the responses of the two local lymph nodes were often observed, suggesting that individual differences in the orientation of the anti‐P815 CTL response do not result from preexisting differences between the animals. Asymmetrical CTL responses persisted in mice that were given a second injection of tumor cells. A possible interpretation of our results is that the major component of the CTL response is made of the progeny of a very small number of CTL precursors that happen to be the first to be stimulated by the tumor antigens.

A number of cytolytic T lymphocyte (CTL) clones derived from several melanoma patients have been found to recognize a majority of melanomas from HLA-A2 patients. We have reported previously that two such CTL clones recognize a product of the tyrosinase gene that is presented by HLA-A2. Here we show that one of these CTL clones recognizes a peptide encoded by the first nine amino acids of the putative signal sequence of tyrosinase. The other CTL clone recognizes a different tyrosinase peptide corresponding to amino acids 368-376. Both peptides contain consensus motifs of HLA-A2 binding peptides.

Cytolytic T lymphocyte (CTL) clones directed against spontaneous mouse leukemia LEC have been obtained. By transfecting a cosmid library into cells which were then tested for their ability to stimulate the CTL, we identified the gene coding for the antigen recognized by one of these CTL clones. It is the gag gene of an endogenous defective retrovirus that belongs to the intracisternal A particle (IAP) family. A gag-encoded nonapeptide presented by the H-2 D-k molecule caused recognition by the anti-LEC CTL clone. Southern blot and polymerase chain reaction analyses indicated that the expression of the antigen by the LEC tumor cell line resulted from the transposition of an IAP sequence into a new genomic location.

It has been reported previously that antitumor cytolytic T lymphocyte (CTL) clones can be isolated from blood lymphocytes of HLA-A2 melanoma patients, after stimulation in vitro with autologous tumor cells, and that some of these CTL clones lyse most HLA-A2 melanomas. A first antigen recognized by such CTL clones was previously shown to be encoded by the tyrosinase gene. We report here the identification of another gene that also directs the expression of an antigen recognized on most melanomas by CTL clones that are restricted by HLA-A2. The gene, designated Melan-A, is unrelated to any known gene. It is 18 kb long and comprises five exons. Like the tyrosinase gene, it is expressed in most melanoma tumor samples and, among normal cells, only in melanocytes.

We have observed delayed-type hypersensitivity (DTH) reactions in immunized mice challenged subcutaneously with class I-binding peptides related to rejection antigens recognized bq cytotoxic T lymphocytes on mutagenized (tum(-)) variants of mastocytoma P815. As observed by skin test in virally infected mice challenged with viral peptides, the intrafootpad injection of tum(-) peptides resulted in a dose-dependent DTH that peaked at approximately 24 h. The response was mediated by CD8(+) cells and could be induced by previous vaccination of mice with live tumor cells, intrasplenic deposition of the eliciting peptide, or adoptive transfer with peptide-pulsed syngeneic dendritic cells. These sensitization procedures resulted in an immunologically specific footpad reaction detectable for up to 2-6 months after priming. The evaluation by DTH in cancer patients of long-lived CD8(+) anti-tumor T cell responses following local challenge with tumor-specific peptides may be of great interest in human immunotherapy trials involving immunization against identified tumor antigens.

To characterize the proteins P91Ap and P198p, of which mutants generate the tum- antigens P91A and P198, respectively, rabbit antisera were raised with ovalbumin-coupled synthetic peptides that correspond to their respective C terminus. In immunoadsorption tests using immobilized protein A the antisera recognized the translation products synthesized by rabbit reticulocyte lysates programmed with the SP6 polymerase transcripts of the P91A and P198 cDNA. The presence of the two proteins was demonstrated by SDS-PAGE and immunoblotting in all the mouse cells and organs examined. P91Ap is a constituent of the cytosol; despite a remarkable homology to the Drosophila diphenol oxidase DOX-A2, it separates from murine catechol oxidase activity in rate zonal sedimentation analysis. P198p is a ribosomal constituent, or a factor firmly linked to both the free and membrane-bound ribosomes. These subcellular localizations strengthen other evidence that the antigens presented to T lymphocytes by class I products of the major histocompatibility complex derive from proteins of the cytosol, or in direct contact with it.

Lymphocytes of melanoma patients can be restimulated in vitro with autologous tumor cells to generate antitumor cytolytic T lymphocytes (CTL). Previous reports have indicated that, when such CTL are obtained from HLA-A2 melanoma patients, they often display broad reactivity on A2 melanoma cell lines. Such antitumor CTL clones, which appeared to recognize the same antigen, were isolated from two patients. We report here the cloning of a cDNA that directs the expression of the antigen recognized by these CTL. This cDNA corresponds to the transcript of the tyrosinase gene. The gene was found to be active in all tested melanoma samples and in most melanoma cell lines. Among normal cells, only melanocytes appear to express the gene. The tyrosinase antigen presented by HLA-A2 may therefore constitute a useful target for specific immunotherapy of melanoma. But possible adverse effects of antityrosinase immunization, such as the destruction of normal melanocytes and its consequences, will have to be examined before clinical pilot studies can be undertaken.

Like many antigens presented by class I molecules the mouse L(d)-binding tum- antigen P91A derives from a cytosolic protein. To decide how stringent this localization is for presentation to cytotoxic lymphocytes (CTL) the P91A template has been inserted in the cDNA of rat esterase ES-10, a protein located in the endoplasmic reticulum (ER), and in the cDNA of mouse interleukin-9, a secretory product of lymphocytes. The esterase construct was also engineered to replace the C-terminal leucine by arginine, which causes secretion of the protein, or to delete the N-terminal presequence, which prevents transfer of the nascent chain to the ER. After cell-free transcription-translation, or transfection in COS cells, the products of the chimeric cDNA had the expected size and localization; however, the truncated form of esterase remained undetected in COS cells. The various chimeric templates were transfected in P1.HTR cells (H-2d); upon challenge with L(d)-restricted anti-P91A CTL the cells were lyzed almost as efficiently as cells transfected with the full-length P91A cDNA. We conclude that peptide fragments that bind to class I molecules of the major histocompatibility complex can be generated in the ER.

Mouse mastocytoma P815 expresses several distinct tumor rejection antigens recognized by syngeneic cytolytic T lymphocytes (CTL). Two of these tumor rejection antigens, P815A and P815B, are encoded by gene P1A, the sequence of which was reported previously. Tumor cell variants having lost one or both of these antigens were isolated by in vitro selection with CTL and also by collecting tumor cells that progressed in vivo after escaping a nearly complete immune rejection process. The structure of gene P1A in these antigen-loss variants was examined. Several A-B- variants presented a complete or partial deletion of the gene. One variant that had lost only antigen A displayed a point mutation in the first exon. Peptides were synthesized that corresponded to the normal sequence located in the region of this point mutation. They sensitized target cells to both anti-A and anti-B CTL. The homologous peptide encoded by the mutated gene of the P815 A-B+ variant sensitized cells only to anti-B CTL. We conclude that anti-A and anti-B CTL recognize on the same peptide two distinct epitopes that are affected differently by the mutation.

The target antigen recognized by H-2K(d)-restricted cytotoxic T lymphocytes (CTLs) specific for a mutagen-induced antigen on DBA/2-derived tumor P815 was identified as the product of a normal cellular gene encompassing a point mutation. Using synthetic peptides, the epitope recognized by these CTLs was narrowed down to be contained within the undecamer KYQAVTTTLEE, incorporating the point mutation. The allele-specific peptide motif for H-2K(d) molecules allowed us to predict the peptide naturally presented by the tumor cells to be the nonamer KYQAVTTTL. Isolation of the natural tum--specific peptide from P198.3 tumor cells and biochemical comparison with the synthetic nonamer confirmed the prediction. This natural nonapeptide is represented by approximately 1 00 copies per tumor cell.

We have reported the identification of human gene MAGE-1, which directs the expression of an antigen recognized on a melanoma by autologous cytolytic T lymphocytes (CTL). We show here that CTL directed against this antigen, which was named MZ2-E, recognize a nonapeptide encoded by the third exon of gene MAGE-1. The CTL also recognize this peptide when it is presented by mouse cells transfected with an HLA-A1 gene, confirming the association of antigen MZ2-E with the HLA-A1 molecule. Other members of the MAGE gene family do not code for the same peptide, suggesting that only MAGE-1 produces the antigen recognized by the anti-MZ2-E CTL. Our results open the possibility of immunizing HLA-A1 patients whose tumor expresses MAGE-1 either with the antigenic peptide or with autologous antigen-presenting cells pulsed with the peptide.

On the basis of the results reviewed here, there are two major mechanisms whereby tumour rejection antigens may arise. The first mechanism is mutational. Point mutations occurring in a large variety of genes may produce new antigenic peptides, either by providing them with the ability to bind to MHC class I molecules or by providing them with a new epitope (Fig. 2). The second mechanism is the activation of a gene that is silent in normal tissues and for which no strong natural tolerance has been established. Plausible candidates for the mutational mechanism are the "tumour specific transplantation antigens" observed on methylcholanthrene induced tumours and tumours induced by ultraviolet light. The diversity of these antigens appears to be very large, like that of the tum- antigens. Moreover, these tumours have been obtained with high doses of carcinogens, which are proven mutagens. On the other hand, a P815 tumour rejection antigen appears to arise through the activation of a silent gene, and it may turn out that this is the rule for most tumour rejection antigens. It is our hope that other genes coding for mouse and human tumour rejection antigens will soon be identified, so that it will become clear whether the activational mechanism is the rule or the exception. In our view, this is a crucial issue. Insofar as tumour rejection antigens result from mutations, they may be highly specific for every individual tumour. The tumour specific nature of these antigens would then be easily ascertained. However, active immunization of cancer patients would require that a tumour cell line be obtained from each patient, a most unpractical prospect. If, on the other hand, production of tumour rejection antigens results from the activation of a normal gene, then there is a good probability that the same gene may be activated in many different tumours, being perhaps preferentially shared by tumours of the same histological type. This would probably not result in the expression of the same antigen in all these tumours, because the patients would differ in their presenting molecules, which are determined by their HLA haplotype. However, a subset of the tumours expressing the same "tumour rejection" gene should share the same class I restricting element, so that all of these patients could be immunized with a cell that would express the gene and carry the appropriate HLA molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

Tumor-associated transplantation antigens (TATA) are commonly found on rodent tumors induced by oncogenic viruses, chemical carcinogens, and ultraviolet (UV) irradiation.1-4 In contrast, spontaneous rodent tumors appear to be incapable of eliciting any rejection response in the syngeneic host.5,6 But further experiments demonstrated that even these tumors express weak TSTA that are recognized by cytolytic T cells (CTL) and are potential targets for immune rejection.7 In man also, there is good evidence that some tumors carry tumor-associated antigens that are recognized by autologous CTL.8 However, it is difficult to evaluate to what extent human tumors carry antigens that can be the targets of an autologous rejection response.What is the molecular nature of TSTA? And what is the relation between their appearance and the tumoral transformation process? These questions are still unanswered because the TSTA, which elicit strong T-cell mediated immune responses, do not stimulate B cells to produce antibodies. It has therefore been impossible to isolate the antigenic molecules by immunoprecipitation. This predicament is not restricted to TSTA: most minor histocompatibility antigens and the male-specific antigen H-Y remain uncharacterized for the same reasons.We have developed a gene transfection approach aimed at identifying directly the genes that code for this type of antigen and we have applied it to the "tum-" transplantation antigens, which are found on mutagenized mouse tumor cells. This methodology also ensured the isolation of the gene coding for a TSTA present on a mouse mastocytoma tumor.